Abstract
Electron Paramagnetic Resonance (EPR) was used to investigate the Tempyo spin label (3-carbamoyl-2,2,5,5-
tetramethyl-3-pyrrolin-1-yloxy) as a report group for the interactions and the conformational changes of lyophilized
bovine serum albumin (BSA) and bovine hemoglobin (BH), as function of pH values in the range 2.5–11. The EPR
spectra are similar with those of other non-covalently spin label porphyrins in frozen solution at very low temperatures.
This behavior indicated a possible spin–spin interaction between the hemic iron and the nitroxide group. The changes
in the EPR spectra as function of the pH are discussed in terms of conformational changes of the proteins. Spectral
simulations and magnetic EPR parameters reveal the following: (i) one single paramagnetic species, with Gaussian
line shape, was used for the best fits of experimental spectra in the case of serum albumin samples; and (ii) a
weighted sum of Lorentzian and Gaussian line shape in the case of hemoglobin samples. The representation of
correlation time vs. pH, reveals a dependence of degree of immobilization of spin label on the conformational changes
of proteins in acidic and basic environment.
SIMONA CAVALU_EPR study of non covalent spin labeled serum albumin and haemoglobin 2002
1. Biophysical Chemistry 99 (2002) 181–188
0301-4622/02/$ - see front matter ᮊ 2002 Elsevier Science B.V. All rights reserved.
PII: S0301-4622Ž02.00182-5
EPR study of non-covalent spin labeled serum albumin and
hemoglobin
S. Cavalu *, G. Damian , M. Dansoreanua, b c
ˆ ¸
University of Oradea, Faculty of Medicine and Pharmacy, Department of Biophysics, RO-3700 Oradea, Romaniaa
‘‘Babes-Bolyai’’ University, Department of Physics, RO-3400 Cluj-Napoca, Romaniab
University of Medicine and Pharmacy ‘‘Iuliu Hatieganu’’, Department of Biophysics RO-3400 Cluj-Napoca, Romaniac
Received 5 February 2002; received in revised form 31 May 2002; accepted 5 June 2002
Abstract
Electron Paramagnetic Resonance (EPR) was used to investigate the Tempyo spin label (3-carbamoyl-2,2,5,5-
tetramethyl-3-pyrrolin-1-yloxy) as a report group for the interactions and the conformational changes of lyophilized
bovine serum albumin (BSA) and bovine hemoglobin (BH), as function of pH values in the range 2.5–11. The EPR
spectra are similar with those of other non-covalently spin label porphyrins in frozen solution at very low temperatures.
This behavior indicated a possible spin–spin interaction between the hemic iron and the nitroxide group. The changes
in the EPR spectra as function of the pH are discussed in terms of conformational changes of the proteins. Spectral
simulations and magnetic EPR parameters reveal the following: (i) one single paramagnetic species, with Gaussian
line shape, was used for the best fits of experimental spectra in the case of serum albumin samples; and (ii) a
weighted sum of Lorentzian and Gaussian line shape in the case of hemoglobin samples. The representation of
correlation time vs. pH, reveals a dependence of degree of immobilization of spin label on the conformational changes
of proteins in acidic and basic environment.
ᮊ 2002 Elsevier Science B.V. All rights reserved.
Keywords: EPR; Spin label; Serum albumin; Haemoglobin
1. Introduction
Proteins are polymeric chains that are built from
monomers called amino acids. All structural and
functional properties of proteins derive from the
chemical properties of the polypeptide side-chain.
The ionizable groups of the side-chains of charged
amino acids are often involved in biochemical
*Corresponding author. Fax: q40-59-41-8266.
E-mail address: scavalu@xnet.ro (S. Cavalu).
transactions (binding, catalysis). Therefore, pH
usually has rather dramatic effects on the function
of proteins. Polar residues are both buried as well
as on the surface of the protein. They either form
hydrogen bonds with other polar residues in the
protein or with water. Non-polar residues do not
interact favorably with water. However, a signifi-
cant number of non-polar residues are also found
on the surface of the protein. Therefore, it is
important to study the interactions of proteins with
well-defined molecules that will confer the total
2. 182 S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188
Fig. 1. The chemical structure of Tempyo spin label.
molecules sufficient affinity for the binding site of
interest on protein w1,2x
The successful application of spin labeling to
protein structure investigations is limited by the
possibility to chemically change specific side
chains in proteins. However, useful information on
protein properties can be obtained by non-covalent
spin labeling if the affinity of the protein for the
label molecules is great enough to affect their
motional freedom w3–6x. The rate of rotation (or
tumbling) of the spin label influences the lineshape
of its EPR spectrum. Therefore, the EPR signal of
a spin label covalently or non-covalently bonded
to a biomolecule can yield a range of information
about its structural environment in conventional
ESR and allows for full spectral coverage w7,8x.
In the same time, EPR has been an invaluable
tool for probing microscopic molecular motions in
a variety of systems, including isotropic solvents
w9x, liquid crystals w10x, model membranes and
biomolecules w11–13x.
In the present work, non-covalent spin labeled
bovine serum albumin (BSA) and bovine hemo-
globin (BH) with Tempyo spin label (3-carba-
moyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) were
investigated both in liquid and lyophilized sam-
ples, in the pH range 2.5–11, in order to obtain
useful information related to the interaction
between the nitroxide group and the functional site
of the proteins.
Interactions of spin label with hemic or non-
hemic proteins might affect the spin label spectra
and in the same time it is well known that the pH
strongly influences the conformation of proteins
leading to significant changes in the type and
degree of these interactions w14x. In this pH range,
we followed the effect of protein conformational
changes on the interactions between the nitroxide
and the functional groups of proteins and also the
pH influence on molecular motion emphasized by
the EPR spectra of the spin label.
2. Materials and methods
Powdered bovine serum albumin and hemoglo-
bin ()95% methemoglobin) from Sigma Chemi-
cals, were used without further purification.
Proteins were hydrated in phosphate buffer physi-
ological saline at a final concentration of 10 M.y3
Tempyo spin-label (3-carbamoyl-2,2,5,5-tetrame-
thyl-3-pyrrolin-1-yloxy), from Sigma Chemicals
(Fig. 1), was added to the liquid samples of each
protein in a final concentration of 10 M (pro-y3
teinyspin label molar ratio 1:1) and the pH values
were adjusted to the desired value in the range
2.5–11. An amount of 5 ml from each sample was
lyophilized for 30 h at y5 8C and used for the
EPR measurement, at room temperature.
EPR spectra for both liquid and lyophilized
samples, were recorded at room temperature with
a JEOL-JES-3B spectrometer, operating in X-band
(9.5 GHz), equipped with a computer acquisition
system. Samples were placed in quartz capillary
tubes. The spectrometer settings were: modulation
frequency 100 KHz, field modulation 1 G, micro-
wave power 20 mW. The computer simulation
analysis of spectra, for obtaining the magnetic
characteristic parameters, was made by using a
program that is available to the public through the
Internet (http:yyalfred.niehs.nihyLMB).
The lineshape of an EPR spectrum depends,
among other factors, on the orientation of the
paramagnetic center with respect to the applied
magnetic field. In a powder, or a frozen aqueous
solution, the paramagnetic centers will be fixed
with a random distribution of orientations, and in
the case of anisotropic g- and hyperfine interac-
tions this will lead to a broadened EPR spectrum,
since all orientations contribute equally. In the
liquid state, however, the paramagnetic centers are
not fixed but undergo rotational fluctuation. In the
case of fast rotation, the anisotropic interactions
are thereby averaged to zero, giving rise to sharp
EPR lines. If the velocity of the rotational motion
3. 183S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188
decreases, the EPR spectrum will approach that of
the powder spectrum. Therefore, a rotational cor-
relation time for a paramagnetic molecule can also
be determined by EPR using semiempirical for-
mula w15x.
For isotropic motion in the rapid tumbling limit,
the spectra will be isotropic with the averages of
the principal components of the g-values and
hyperfine splitting factor, a . The rate of theN
isotropic motion determines the relative widths of
resonances and the width, DH , of an individualm
(hyperfine) line, in the first approximation can be
written as a function of the z component of the
nitrogen nuclear spin number (msy1, 0, 1)
w16,17x:
2
DH sAqBØmqCØm (1)m
where the A coefficient includes other contribu-
tions than motion. The terms B and C are functions
related to the rotational correlational time (t) and
can be defined as a function of peak-to-peak line
width of the central line, DH wGx, and the ampli-0
tudes of the mth line I w14x:m
B E
1 I I0 0
C FBs DH y0 y y2 I ID G1 y1
N
ws0.103v DgDae
w z3N 2 2 y1x |xŽ .Ž . Ž .q3 dg da t 1q 1qv t (2)B e B
y ~4
B E
1 I I0 0
C FCs DH q y20 y y2 I ID G1 y1
w 36 N N2 2 xŽ . Ž .s1.181Ø10 Da q3 da t 1yw x c
y 8
z12 2 2 2y1 y1|Ž . Ž .= 1qv t y 1qv t (3)c c e c
~8
in which
1 1N N N N N N N
Ž . Ž .Da sa y a qa , da a ya (4)zz xx yy xx yy
2 2
1 1
Ž . Ž .Dgsg y g yg , dgs g yg (5)zz xx yy xx yy
2 2
and v s8.8=10 Na M, a is the isotropicy6 N N
N
hyperfine splitting and v the ESR spectrometere
frequency in angular units.
In the range from 5=10 to 10 s (motiony11 y9
in the rapid tumbling limit) and magnetic field
above 3300 G, Dg and Da vanish, and theN
correlation times t and t are directly related toB C
the B and C coefficients by the following simple
relations w5x:
t st sK ØB (6)B z 1
t st sK ØC (7)C x,y 2
where K s1.27=10 and K s1.19=10 . They9 y9
1 2
average correlation times is:
1y2
Ž .ts t Øt (8)B c
The slow motion of spin probe, lead to a
broadening of the EPR lines. In this case, the
rotational correlation time, t, is larger than 10y9
s, and thus, the relation (8) is not applicable.
The isotropic nitrogen hyperfine splitting chang-
es to a powder like spectrum, with the peak-to-
peak distance between the external peaks of the
spectrum w2Øa9 (N)x depending on the magnitudezz
of the rotational correlation time, t w18,19x. Anoth-
er lineshape theory for slow isotropic Brownian
rotational diffusion of spin-labeled proteins has
been developed by Freed w9x. Thus, the correlation
time can be evaluated from the ratio of the
observed splitting between the derivate extreme
a9 and principal value a , determined from rigidzz zz
matrix spectrum w5,7x:
bB E
a9zz
C Ftsa 1y (9)
aD Gzz
The a and b parameters depend on the kind of
the diffusion process, where a and b are empirical
constants, which are tabulated in e.g. Poole and
Farach (1987) w15x. For small spin probe, the
intermediate jump diffusion is preferable w15x.
3. Results and discussion
In the low concentration liquid state BSA and
BH solution, the Tempyo spin label which is a
relatively small molecule, gives rise to a spectrum
with narrow lines and constant hyperfine splitting,
typical for fast isotropic rotational motion (Fig. 2)
with a very low rate of migration between protein
molecule and water. For this kind of rotation, the
4. 184 S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188
Fig. 2. EPR spectrum of the Tempyo spin label at low pH.
Fig. 3. Experimental (——) and simulated (««) EPR spectra
of Tempyo spin label in lyophilized bovine serum albumin at
various pH values.
Table 1
Magnetic parameters values of Tempyo spin label in lyophili-
zed bovine serum albumin at various pH values
pH gxx gyy gzz A (G)xx A (G)yy A (G)zz
11 2.01028 2.00841 2.00441 4.72 8.53 35.48
9.5 2.00113 2.00762 2.00484 6.16 5.91 33.96
8.1 2.01128 2.00781 2.00461 6.41 5.69 32.98
6.7 2.00113 2.00742 2.00500 6.15 6.55 32.20
5.5 2.01147 2.00809 2.00505 6.03 7.83 35.45
4.5 2.01259 2.00672 2.00588 7.79 6.98 35.52
3.5 2.01243 2.00660 2.00538 8.28 5.34 35.46
2.5 2.01269 2.00662 2.00459 6.38 5.66 35.91
rotational correlation time can be estimated from
the intensity ratio of the low-field and high-field
N-lines using a semi-empirical formula (8). For
the Tempyo spin label in BSA and BH aqueous
solution, the calculated rotational correlation times
was 2=10 s and 2.5=10 , respectively,y10 y10
which is consistent with fast rotation as expected
for a small molecule.
No detectable changes were observed in EPR
spectra of aqueous solution of Tempyo spin label
without proteins, at different pH values.
The characteristic EPR spectra of a Tempyo spin
label in lyophilized BSA is due primarily to
anisotropy in the nitrogen hyperfine coupling
which is typical for slow rotation. For slow rota-
tions, the EPR spectrum of spin labels depends, in
a much more complicated fashion, on the com-
bined influences of molecular motion and magnetic
interactions. Fig. 3 displays the experimental and
simulated spectra for Tempyo spin label in lyo-
philized BSA at different pH values. In order to
find the magnetic parameters, the experimental
EPR spectra were simulated. The best fit of exper-
imental EPR spectra of Tempyo spin label in
lyophilized BSA, was obtained assuming a single
paramagnetic species with magnetic parameters
listed in Table 1, and a Gaussian lineshape corre-
sponding to static dipolar interactions.
The main feature of the EPR spectra of Tempyo
spin label in lyophilized haemoglobin (Fig. 4),
like the Tempyo spin label in lyophilized BSA,
exhibit the characteristics to slow motion of spin
label but with more broadening of the lineshape.
The best fit of the simulated experimental EPR
spectra can be obtained by assuming the presence
of two functional groups in hemoglobin associated
with two non-equivalent paramagnetic species.
Computer simulations indicate a weighted sum of
mixed Gaussian lineshapes (static dipolar interac-
tions) and Lorentzian lineshapes (spin–spin inter-
actions). Generally, the broadening of the Gaussian
lineshape are due to static dipolar interactions of
5. 185S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188
Fig. 4. Experimental (——) and simulated (««) EPR spectra
of Tempyo spin label in lyophilized bovine hemoglobin at var-
ious pH values.
Fig. 5. The experimental EPR spectrum and its subspectra of
Tempyo spin label in lyophilized hemoglobin at pH 2.5
the spin label molecules whereas the broadening
of the Lorentzian lineshape is due to to spin–spin
interactions w20x. Figs. 5 and 6 present the EPR
spectra of Tempyo spin label in the lyophilized
BH at low and high pH, in which the contribution
of each species is displayed. At low pH (pH 2.5),
the main contributions are due to species with
Gaussian line shape (;80%). This contribution
decreases to ;50% at high pH (pH 11). The first
species with Gaussian lineshape and poor resolved
hyperfine splitting is not influenced by the pres-
ence of the hemic iron and, therefore, we assume
to be located far from the hemic group. The second
species with Lorentzian lineshape and a well
resolved hyperfine structure is located, probably,
near the hem group, giving rise to a spin–spin
interaction between the nitroxide radical and the
paramagnetic iron of the hem group. Our results
are in accordance with covalently labeled methe-
moglobin and other porphyrins in frozen samples
under 50 K w21,22x. In these previous studies,
spectra of covalently labeled methemoglobin were
analyzed by using perturbation calculations in
order to estimate the iron to nitroxyl distances and
it was suggested that plausible distances are in the
range of 14.5–17.5 A. The g tensor and A tensor˚
components used in the simulation for the best fit
values of the simulation of the effective powder
spectrum, are presented in Table 2.
Due to increased spatial restrictions of protein
structure in the vicinity of the label, by lyophili-
zation, the mobility of spin label is slow on the
EPR time scale (;5=10 s ), leading to a6 y1
broadening of the EPR lines, with the peak-to-
peak distance between the external peaks of the
spectrum w2Øa9 (N)x depending on the magnitudezz
of the rotational correlation time, t. Generally,
broadening of the peaks in an EPR spectrum is
6. 186 S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188
Fig. 6. The experimental EPR spectrum and its subspectra of
Tempyo spin label in lyophilized hemoglobin at pH 11.
Table 2
Magnetic parameters values of Tempyo spin label in lyophili-
zed bovine hemoglobin at various pH values
pH gxx gyy gzz A (G)xx A (G)yy A (G)zz
11 2.01242 2.00771 2.00372 4.81 8.70 34.01 L( )
2.01117 2.00497 2.00610 6.42 7.76 33.16 G( )
9.5 2.00935 2.00839 2.00478 6.19 13.89 34.82 L( )
2.01079 2.00695 2.00465 6.11 7.29 33.06 G( )
8.1 2.01133 2.00702 2.00464 8.38 6.68 33.87 L( )
2.01500 2.00428 2.00592 8.64 6.50 32.60 G( )
6.7 2.00115 2.00773 2.00425 3.27 8.20 33.73 L( )
2.01213 2.00476 2.00556 7.43 8.88 35.84 G( )
5.5 2.012442 2.00821 2.00471 9.52 7.32 34.62 L( )
2.001050 2.00592 2.00541 5.86 6.12 35.45 G( )
4.5 2.01609 2.00768 2.00522 9.20 7.66 36.03 L( )
2.01198 2.00416 2.00523 7.43 8.88 35.15 G( )
3.5 2.01265 2.00816 2.00413 4.14 5.39 36.59 L( )
2.01169 2.00287 2.00506 7.06 9.36 35.46 G( )
2.5 2.01344 2.00844 2.00506 9.83 10.38 36.26 L( )
2.01270 2.00471 2.00548 4.67 5.80 35.78 G( )
(L), Lorentzian lineshape; (G), Gaussian lineshape.
Fig. 7. Correlation times (t) as a function of pH for Tempyo
spin label in lyophilized bovine serum albumin (j) and bovine
hemoglobin (m).
indicative of immobilization of the spin label,
whereas sharpening of the peaks is indicative of
an increase in label mobility. By comparing the
apparent nitrogen hyperfine splitting wtermed a9zz
(N)x with the nitrogen hyperfine splitting obtained
from their rigid limit values wa (N)x, the rotation-zz
al correlation times can be calculated using for-
mula 9. The values of a and b coefficients depend
on the motional model. The study of the influence
of different diffusional models on the spectral line
shape in the regime of slow motional spin label
by high EPR fields has shown that jump diffusion
mainly affects the line widths at the same motional
rates w23x. In our calculations, the intermediate
jump diffusion model was considerate, with coef-
ficients values of as5.4=10 s and bsy1.36y10
w15x. The average correlation time for different
values of pH are plotted in Fig. 7. As shown in
figure, the pH influences the rotational correlation
time. In the acidic pH range the NH groups of2
the label molecule as well as those of the amino
acids residues are protonated. The fact that t shows
greater values in this range followed by a signifi-
cant decrease in the basic pH range, indicate a
low mobility of spin label in acidic environment
while an increasing of mobility can be noticed in
the basic pH range. A minimum mobility can be
observed around the isoelectric point of both pro-
teins (for BSA pH s4.8, and for BH, pH s6.8).i i
From the pH dependence of correlation time
(involving the mobility of the label as well), we
assume that in an acidic environment the mobility
of spin label molecules are reduced due to the
7. 187S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188
formation of hydrogen bonds between the NH2
group of spin label and side chains of neighboring
amino acids. One can correlate this observation
with the fact that serum albumin undergoes revers-
ible isomerization in the pH range 2.7–7 from the
expanded form characterized by 35% a-helix con-
tent, to normal form characterized by 55% a-helix
content accompanied by a decrease in b-sheet
w24,25x. It is well known that b-sheet conformation
favors the formation of hydrogen bonding.
By comparing with the BSA case, we notice
that the mobility of Tempyo is greater with respect
to hemoglobin, which is not surprising if we take
into account that hydrogen bonding opportunities
depends on the b-sheet content: in hemoglobin the
b-sheets represent 50% while in BSA the percent-
age varies from 70 to 45%, depending on the pH.
As shown in Fig. 7 the mobility of this species
increases in an acidic environment, reaching a
maximum value corresponding to pH 6.8 followedi
by a slow decrease. We suggest that in basic pH
range, where the label is not subject to strong
electrostatic interactions, dipolar and spin–spin
interactions are manifested almost with the same
contribution in the broadening of the spectrum.
4. Conclusions
EPR spectroscopy is very useful to study the
mobility of nitroxide radicals with respect to hemic
or non-hemic proteins in different environmental
conditions. Non-covalent labeling of proteins can
give valuable information on the magnetic inter-
actions between the label molecule and the para-
magnetic center of the proteins. The relevance of
this interaction can be obtained from lineshape
analysis: computer simulations for non-hemic pro-
tein assume a Gaussian lineshape, while for hemic
protein a weighted sum of Lorentzian and Gaussian
components is assumed. The contribution of the
each line shape to experimental spectrum depends
on the pH.
References
w1x T.E. Creighton, Proteins: Structures and Molecular Prop-
erties, W.H. Freeman and Company, New York, 1983.
w2x J.R. Foster, in: V.M. Rosenoer, M. Oratz, M.A. Roths-
child (Eds.), Albumin Structure, Function and Uses,
Pergamon, Oxford, 1977.
w3x J.D. Morrisett, R.W. Wien, H.M. McConnell, The use
of spin labels for measuring distances in biological
systems, Ann. N.Y. Acad. Sci. 222 (1973) 149–162.
w4x P. Jost, O.H. Griffith, Electron spin resonance and the
spin labeling method, in: C. Chignell (Ed.), Methods in
Pharmacology, Appleton, New York, 1972.
w5x J.D. Morrisett, H.J. Pownall, A.M. Gotto, Bovine serum
albumin, study of the fatty acid and steroid binding
sites using spin labeled lipids, J. Biol. Chem. 250 (1975)
2487–2494.
w6x J.D. Morrisett, Spin labeled enzymes, in: J. Berliner
(Ed.), Spin Labelling—Theory and Application, Aca-
demic Press, 1975.
w7x P. Frajer, Electron spin resonance spectroscopy labeling
in peptide and protein analysis, in: R.A. Meyers (Ed.),
Encyclopedia of Analytical Chemistry, John Wiley &
Sons Ltd, 2000.
w8x R. Biswas, H. Kuhne, G.W. Brudvig, V. Gopalan, Use
of EPR spectroscopy to study macromolecular structure
and function, Sci. Prog. 84 (1) (2001) 45–68.
w9x J.S. Hwang, R.P. Mason, L.-P. Hwang, J.H. Freed,
Electron spin resonance studies of anisotropic rotational
reorientation and slow tumbling in liquid and frozen
media. III. Perdeuterated 2,2,6,6,-tetramethyl-4-piperi-
done N-oxide and an analysis of fluctuating torques, J.
Phys. Chem. 79 (1975) 489–511.
w10x E. Meirovitch, D. Igner, G. Moro, J.H. Freed, Electron-
spin relaxation and ordering in smectic and supercooled
nematic liquid crystals, J. Chem. Phys. 77 (1982)
3915–3938.
w11x H. Tanaka, J.H. Freed, Electron spin resonance studies
on ordering and rotational diffusion in oriented phos-
phatidylcholine multilayers: evidence for a new chain-
ordering transition, J. Phys. Chem. 88 (1984)
6633–6643.
w12x D. Marsh, L.I. Horvath, Spin label studies of structure
and dynamics of lipids and proteins in membranes, in:
A.J. Hoff (Ed.), Advance EPR—Application in Biology
and Biochemistry, Elsevier, Amsterdam, 1989.
w13x S. Schreier, C.F. Polnaszek, I.C.P. Smith, Spin labels in
membranes, Biochim. Biophys. Acta 515 (1978)
375–436.
w14x G.R. Eaton, S.S. Eaton, Interaction of spin labels with
transition metals, Co-ordinat. Chem. Rev. 26 (1978)
207–262.
w15x C.P. Poole, H.A. Farach, Theory of Magnetic Reso-
nance, John Wiley & Sons, New York, NY, 1987, pp.
319–321.
w16x A. Redfield, The Theory of Relaxation Processes’, Adv.
Magn. Reson. 1 (1965) 1.
w17x S.A. Goldman, G.V. Bruno, C.F. Polnaszek, J.H. Freed,
An ESR study of anisotropic rotational reorientation
8. 188 S. Cavalu et al. / Biophysical Chemistry 99 (2002) 181–188
and slow tumbling in liquid and frozen media, J. Chem.
Phys. 56 (1972) 716–735.
w18x D.J. Schneider, J.H. Freed, in: L.J. Berliner, J. Reuben
(Eds.), Spin Labeling Theory and Applications, Plenum
Press, New York, 1989, pp. 1–76.
w19x D. Marsh, in: L.J. Berliner, J. Reuben (Eds.), Spin
Labeling Theory and Applications, Plenum Press, New
York, 1989, pp. 255–303.
w20x L.J. Berliner, Spin Labeling, Theory and Application,
Adademic Press, New York, NY, 1976.
w21x V. Budker, J.-L. Du, M. Seiter, G.R. Eaton, S.S. Eaton,
Electron–electron spin–spin interaction in spin labeled
low-spin methemoglobin, Biophys. J. 68 (1995)
2531–2542.
w22x M.H. Rakowsky, M.K. More, A.V. Kulikov, G.R. Eaton,
S.S. Eaton, Time-domain electron paramagnetic reso-
nance as a probe of electron–electron spin–spin inter-
action in spin-labeled low-spin iron porphyrins, J. Am.
Chem. Soc. 117 (1995) 2049–2057.
w23x K.A. Earle, D.E. Budil, J.H. Freed, 250-GHz EPR of
nitroxide in the slow-motional regime: models of rota-
tional diffusion, J. Phys. Chem. 97 (1993)
13289–13297.
w24x J.F. Foster, in: V.M. Rosenoer, M. Oratz, M.A. Roths-
child (Eds.), Albumin Structure, Function and Uses,
Pergamon, Oxford, 1977, pp. 53–84.
w25x D.C. Carter, J.X. Ho, Structure of bovine serum albu-
min, Adv. Protein. Chem. 45 (1994) 153–203.